West Nile virus (WNV), mosquito-borne member of the Flaviviridae family was first introduced in 1999 epidemic and quickly spread throughout the US. After a brief decline in WNV infections, there was a sudden surge in 2012, the largest seen in the US, with 5,387 cases in 48 states with 243 deaths (CDC). Of these, 2,734 (51%) were classified as neuroinvasive disease (CDC). WNV infections in humans are generally asymptomatic or exhibit mild flu-like symptoms. However, in some hospitalized patients, WNV infections lead to severe neurological sequelae resulting in higher percent of mortality. Extensive research has shed light on the biology and pathogenesis of WNV. However, currently, there are no vaccines or antiviral drugs available for human use. This multi-disciplinary R21 proposal addresses this critical need. Using high throughput screen (HTS), we have identified five lead compounds with IC50 values in the low to submicromolar range inhibiting both WNV and dengue virus proteases. The strengths of our proposal are: (1) Our lab was the first to establish a sensitive fluorescence-based in vitro assay for dengue virus (DENV) protease which is now adopted in various HTS campaigns by several groups. (2) Our HTS campaign of ~120,000 compounds at NSRB at Harvard Medical School resulted in 73 drug-like compounds representing five distinct chemical scaffolds that exhibited 51-90% inhibition of DENV2 and WNV proteases at 10 mM. (3) The five selected compounds from this list all have molecular weights ranging between 338-392 Da, cLogP: 1.8-4.4, and ligand efficiency: 0.25-0.32. These values are well within Lipinski's rule of five. (4) Since the lead compounds were found to inhibit both WNV and all four serotypes of DENV proteases, optimization of one or more of these compounds could lead to broad spectrum inhibitor(s) for related viruses. We propose the following Specific Aims. In Aim 1.1, we propose to optimize the two of the hits identified in the HTS and selected for this proposal into potent WNV and DENV2 protease inhibitors by iterative medicinal chemistry, biochemical virology, molecular modeling and X-ray crystallography. The potencies (IC50s) of the derivatives of the two lead compounds will be determined in inhibition of the viral protease in vitro and a structure activity relationship (SAR) will be established. In im 1.2, we propose to optimize the WNV and DENV2 protease expression systems in E. coli to produce mg quantities of purified proteases suitable for crystallization and determine the structures of ligand-bound complexes. In Aim 2, we propose to determine the EC50 values, the efficacy of these compounds in inhibition of WNV and DENV2 replication by 50% using WNV and DENV2 Renilla luciferase (Rluc) reporter replicon-expressing monkey kidney (Vero) and BHK-21 cells, respectively. In addition, we propose to validate EC50 values for two compounds using WNV Reporter replicon Virus-like Particles (RVPs) and infectious WNV in collaboration with Dr. Ted Pierson (NIAID) in the BSL-3 facility. The cytotoxicity (CC50) of the lead compounds which show the greatest potencies (IC50 and EC50 values) will be determined. In the R21 phase, we propose to achieve the following milestone: We will identify 1-3 compounds with IC50 and EC50s values in the range of ~500 nM in inhibiting WNV and DENV2 viral replication in Vero and BHK-21 cells with low cytotoxicity (e 200 mM). We will perform P450 inhibition, mouse metabolic stability, and Caco-2 cell permeability for the two chosen lead compounds in Year 1 and for the five optimized compounds in year 2. In addition, we will perform plasma protein binding for the five compounds and mouse PK studies for the two of the most promising compounds. As a future goal (beyond the R21 phase), Two optimized compounds that have desirable drug-like characteristics will be tested in a mouse model for WNV through collaboration with Dr. Phil Murphy, M.D., Chief, Laboratory of Molecular Immunology, NIAID/NIH as well as in the AG129 mouse model for dengue virus in collaboration with Dr. Sujan Shresta, La Jolla Institute of Allergy and Immunology, La Jolla, CA.